Macrophage‐targeted delivery of siRNA to silence Mecp2 gene expression attenuates pulmonary fibrosis

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by the infiltration of macrophages in the fibrotic region. Currently, no therapeutic strategies effectively control disease progression, and the 5‐year mortality of patients after diagnosis is unacceptably high. Thus, developing an effective and safe treatment for IPF is urgently needed. The present study illustrated that methyl‐CpG‐binding protein 2 (MECP2), a protein responsible for the interpretation of DNA methylome‐encoded information, was abnormally expressed in lung and bronchoalveolar lavage fluid samples of IPF patients and mice with onset of pulmonary fibrosis. And further studies verified that the overexpression of MECP2 occurred mainly in macrophages. Inhibition of Mecp2 expression in macrophages robustly abrogated alternatively activated macrophage (M2) polarization by regulating interferon regulatory factor 4 expression. Accordingly, cationic liposomes loading Mecp2 small interfering RNA (siRNA) were raised for the treatment of pulmonary fibrosis. It was noted that the liposomes accumulated in the fibrotic region after intratracheal injection, especially in macrophages. In addition, intratracheal administration of Mecp2 siRNA‐loaded liposomes significantly reversed the established pulmonary fibrosis with few side‐effects and high safety coefficients. Collectively, these results are essential not only for further understanding the DNA methylation in pathogenesis of IPF but also for providing a potent therapeutic strategy for IPF treatment in the clinic practice.


| INTRODUCTION
Idiopathic pulmonary fibrosis (IPF) is a progressive, lethal fibrotic lung disease with unknown etiology. 1 Although pirfenidone and nintedanib are approved by the FDA and modify disease progression in some patients, IPF still carries a poor prognosis, with a median survival of 3.8 years among adults 65 years of age or older. 1 Therefore, it is necessary to develop safe and effective therapeutic strategies for IPF that can be used in clinical practice.
Macrophages are the most abundant immune cells in normal lungs (~70% of immune cells) and are characterized by their plasticity. 2 These cells are activated by Th1 cytokines and/or microbial agents to exhibit a classically activated phenotype (M1) or by Th2 cytokines to exhibit an alternatively activated phenotype (M2). 3 A growing body of evidence supports that macrophages actively participate in the pathogenesis of IPF. 2,4 Specifically, these cells produce a myriad of profibrotic mediators, such as transforming growth factorbeta 1 (TGF-β1), platelet-derived growth factor (PDGF), and found in inflammatory zone 1 (Fizz1), to promote fibroblast differentiation, proliferation and migration, leading to high levels of extracellular matrix (ECM) deposition in the lung parenchyma, impaired functional gas exchange, respiratory failure, and even death. 2,5,6 Indeed, modulating M2 macrophage polarization is a feasible strategy for the treatment of pulmonary fibrosis. 7 DNA methylation is a bridge between environmental stimuli and gene expression. Recent studies have revealed that DNA methylation is involved in the pathogenesis of IPF. 8,9 Methyl-CpG-binding protein 2 (Mecp2), a member of the methyl-CpG-binding domain (MBD) protein family, is responsible for the interpretation of DNA methylome-encoded information. 10 In general, Mecp2 directly binds to methylated CpG DNA and then recruits other remodelers or enzymes to form a complex, leading to the repression or activation of gene expression. 11 There is weak evidence that Mecp2 is involved in the pathogenesis of pulmonary fibrosis. 12 However, the exact mechanism has yet to be fully elucidated. Our previous study illustrated that DNA methylation participates in M2 macrophage polarization. 13 Interestingly, aberrant expression of MECP2 was detected in the lung and bronchoalveolar lavage fluid (BALF) samples of IPF patients and mice with pulmonary fibrosis, and this aberrant expression occurred primarily in macrophages. These observations prompted us to hypothesize that MECP2 might orchestrate M2 macrophages polarization during the development of IPF. Herein, we provided convincing evidence that silencing Mecp2 expression alleviated the M2 program by regulating interferon regulatory factor 4 (Irf4) expression.
RNA interference (RNAi)-based therapy has emerged as a promising therapeutic strategy in chronic diseases due to its excellent ability to silence gene expression in a highly sequence-specific manner. 14 In general, RNAi delivers specific small interfering RNA (siRNA) to target tissues and/(or) cells with fewer side-effects and more effectiveness than traditional therapies. 15 In the current study, we showed that Mecp2 siRNA-loaded liposomes specifically targeted macrophages and passively accumulated in the pulmonary fibrotic areas of mice with pulmonary fibrosis following intratracheal injection. Notably, treatment with Mecp2 siRNA-loaded liposomes robustly reversed established pulmonary fibrosis in a macrophage dependent manner.
Collectively, our data suggest that Mecp2 is essential for the progression of pulmonary fibrosis, and therefore, intratracheal injection of Mecp2 siRNA-loaded liposomes could be a viable therapeutic approach for pulmonary fibrosis. Antibodies against CD68, CD206, and TGF-β1 were purchased from Santa Cruz Biotechnology. Antibodies against arginase-1 and fibronectin were purchased from Abcam. Antibodies against Mecp2, IRF4, inducible nitric oxide synthase (iNOS), and α-SMA were purchased from Cell Signaling Technology. Antibodies against collagen I, Irf4, Gapdh, and β-actin were purchased from Proteintech, and antibodies against Ym1 were purchased from Thermo Fisher Scientific. APC-conjugated anti-mouse F4/80, PE-conjugated anti-mouse CD11c, and FITC-conjugated antimouse CD206 antibodies were purchased from BioLegend.

| Human samples
Human lung tissues were collected from patients with non-small cell lung cancer (n = 5) and IPF (n = 5). BALF was collected from healthy volunteers and IPF patients at Tongji Hospital, followed by informed consent. The diagnosis of IPF was made according to the American Thoracic Society/European Respiratory Society consensus diagnostic criteria. 16

| Animals studies
Eight-week-old male C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were maintained under specific pathogen-free conditions. Induction of pulmonary fibrosis in C57BL/6 mice was performed by intratracheal injection of BLM (2 U/kg) or phosphate-buffered saline (PBS; as a control) with a high pressure atomizing needle (Cat: BJ-PW-M; Bio Jane Trading Limited) after anesthetizing with pentobarbital sodium (60 mg/kg). For the therapeutic experiments, the mice were adminis-

| Western blotting analysis
Mouse and human lung tissues were homogenized in RIPA lysis buffer (Beyotime) containing a protease inhibitor cocktail (Roche), and equal amounts of lysates were separated on 10% polyacrylamide gels (Sigma-Aldrich) and transferred onto polyvinylidene difluoride membranes as previously described. 17 Target protein analysis was performed as described using appropriate primary antibodies, followed by probing with the corresponding horseradish peroxidase-conjugated secondary antibodies. The reactive bands were visualized using ECL reagents (Servicebio), and the band intensities were analyzed using ImageJ software.

| Histological and immunofluorescence staining
Human lung tissue and the left lung of mice were inflated in fresh 4% paraformaldehyde for 24 h at room temperature. Then, the lung tissue was embedded in paraffin and sliced into 5 μm sections. The sections were subjected to hematoxylin and eosin (H&E), Sirius red and Masson's trichrome staining. The Ashcroft scores of the mice were determined to assess the severity of lung fibrosis in each mouse by a blinded observer according to the established protocol. 18 For immunofluorescence staining, BALF cytospin slides or paraffin sections were probed with antibodies against CD68, MECP2, and IRF4, followed by staining with Alexa Fluor 594-labeled anti-mouse/rabbit or Alexa Fluor 488-conjugated anti-rabbit/mouse antibodies (Invitrogen).

| Cell culture
Primary bone marrow-derived macrophages (BMDMs) were isolated and differentiated with M-CSF as previously described. 19 BMDMs were cul-

| RT-PCR
Total RNA was isolated from the mouse lung and cultured cells with the TRlzol reagent (Takara). The RNA quantity and quality were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).
cDNA was prepared using a cDNA synthesis kit (Takara). Quantitative RT-PCR analysis was performed using SYBR Premix Ex Taq (Takara) as previously described. 20 The primer sequences for the target genes are listed in Table 2. All reactions were performed in triplicate.

| Preparation of siRNA-loaded liposomes
siRNA-loaded liposomes were constructed as previous described. 6,21 Briefly

| Hydroxyproline level analysis
The lung hydroxyproline level was measured with a hydroxyproline assay kit from Nanjing Jiancheng Institute of Biotechnology as previously described. 22 Briefly, the fresh lung tissues were weighed and alkaline hydrolyzed for 20 min at 100 C. After adjusting pH to 6.0-6.8, the hydrolysates were refined with active carbon and centrifuged at 3500 rpm for 10 min. The supernatants were then undergone a series of chemical reactions, and finally OD values were determined at 550 nm using a microplate reader (ELx800; BioTek Instruments, Inc.).
The hydroxyproline content in the lung tissue was given as μg hydroxyproline per mg lung tissue by comparing with the hydroxyproline standard.

| Statistical analysis
Comparisons between groups were undertaken using the Prism soft-  BMDMs after LPS treatment ( Figure S2).
It is well known that IL-4/STAT6 signaling is critical for optimal and sustained macrophage M2 polarization upon IL-4 stimulation. 23 We then compared changes in the levels of p-STAT6 between Mecp2 siRNA-or Scr siRNA-treated BMDMs following IL-4 stimulation for 1 h. However, no significant difference in p-STAT6 was detected between Mecp2 siRNA-or Scr siRNA-treated BMDMs ( Figure S3).
Given that Mecp2 could repress the transcriptional activity of Pu.1, 24 and Pu.1 directly binds to the Irf4 promoter and suppresses its expression, 25 we hypothesized that Mecp2 could exacerbate M2  Figure S4). In addition, the obtained liposomes were able to encapsulate siRNA with a high entrapment efficiency of over 95% and the loading efficiency was around 19.8 ± 1.7% ( Figure S4). Subsequently, we evaluated the morphology of the prepared liposomes by TEM. siRNA-loaded liposomes demonstrated a core-shell structure with a well-defined spheroidal shape (Figure 4b).
The light-colored circles around the liposomes represented the lipid layer and darker core was the hydrophilic part inside liposomes sta-  (Figure 4i). Given that some kinds of nanoparticles could induce severe alveolar injury, we next examined the toxicity of liposomes in vitro and vivo to address this concern. Interestingly, the liposomes showed favorable biocompatibility which was verified by CCK8 assay ( Figure S5a). In addition, intratracheal injection of siRNA-loaded liposomes seemed to be safe, as no obvious alveolar damage of other organs injury was detected ( Figure S5b,c). DNA methylation is an epigenetic mechanism involving the addition of a methyl group from S-adenosyl methionine to adenine or cytosine to form N 6 -methyladenine, 5-methylcytosine, and N 4methylcytosine. 27 Generally, DNA methylation in the promoter region of a gene represses its transcription. There is compelling evidence that DNA methylation is involved in the pathogenesis of IPF. 28    However, the underlying mechanism by which Mecp2 regulates Irf4 expression still needs further study.
To translate these observations into clinical treatments, we focused on RNAi-based therapy. 15 At present, the main challenge for implementing siRNA therapies into clinical practice is the lack of effective vectors that protect the siRNA against nuclease degradation and deliver it to target cells without severe side-effects. 36 Herein, we

| CONCLUSION
In this study, we demonstrate compelling evidence indicating that

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.